BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages 48]-486

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1 Vol. 41, No. 3, March 1997 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages 48]-486 INACTIVATION OF ACONITASE IN YEAST EXPOSED TO OXIDATIVE STRESS Keiko Murakami and Masataka Yoshino* Department of Biochemistry, Aichi Medical University, , Japan Received November 14, 1996 Nagakute, Aichi SUMMARY Inactivation of aconitase by oxidative stress was analyzed under the in vivo and in si~u conditions of yeast cells. Treatment of yeast cells with paraquat caused a specific inactivation of aeonitase without affecting the activity of other citric acid cycle enzymes. Addition of copper plus aseorbic acid to permeabilized yeast cells also inactivated aconitase, but did not affect other TCA cycle-related enzymes. Inactivation of aconitase was suggested to be due to the superoxide and hydroxyl radicals produced from the reaction of 02 with paraquat and by Fenton reaction with copper and ascorbic acid under the in vivo and in situ conditons of yeast, respectively. Citrate the substrate of aconitase effectively protected aconitase from the oxidative inactivation. Toxicity of oxygen to yeast cells can be explained by the specific inactivation of aconitase by oxygen radicals. Increased concentrations of citrate can act as a defense mechanism against oxidative inactivation of aconitase under the exposure of aerobically grown yeast to oxidative stress. Key words: Aconitase, Oxygen toxicity, Superoxide, Paraquat, Hydroxyl radical, Yeast INTRODUCTION Oxidative stress results from the production and accumulation of reactive oxygen species such as superoxide anion (02" -), hydrogen peroxide (H202), and hydroxyl radical (HO'). These reactive oxygen species are capable of oxidizing, and therefore damaging, cellular components including DNA, proteins, and membranes (i). Most eukaryotes and prokaryotes respond to oxidative stress: growth of the yeast mutant deficient in superoxide dismutase (EC ) is markedly inhibited by the presence of oxidants (2), suggesting that the superoxide radical is an important agent in the mediation of oxygen toxicity (3, 4). Recent studies showed that most sensitive target of the superoxide radical is aconitase (EC ) *To whom correspondence should be addressed. FAX: (0561) yoshino@amugw.aichi-med-u.ac.jp /97/ /0 Copyright by Ac~Memic Press Australia. All riffhts of reproduction in any Jbrm reserved.

2 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL containing prosthetic iron-sulfur clusters; the citric acid enzyme in bacteria (5) and mammalian cells (6). We now report that the target of oxidative stress on yeast is aconitase under the in vivo and -iin situ conditions: aconitase was specifically inactivated by an exposure of yeast cells to paraquat, and by addition of copper and ascorbic acid. Protection by citrate of oxidative inactivation of aconitase was discussed as a defense mechanism of aerobically grown yeast against oxidative stress. MATERIALS AND METHODS Materials. The sources of materials in this work as follows: NADP- and NAD-isocitrate dehydrogenases from. Boehringer-Mannheim-Yamanouchi (Tokyo, Japan), threo-ds-isocitrate and paraquat (methyl viologen) from Sigma (Tokyo, Japan), and 3-(N-Morpholino)propanesulfonic acid (Mops) from Dojindo Co. (Kumamoto, Japan). Baker's yeast was purchased locally. Determination of enzyme activity. Activities of NADP- (EC ) and NAD-linked isocitrate dehydrogenase (EC I.i.I.41), and malate dehydrogenase (EC ) were determined by following the change in absorbance at 340 nm at 37~ Assay mixtures were essentially similar to those described previously (7). Fumarase (EC ) activity was determined by the formation of fumarate from malate (8). Aconitase was assayed by the coupled assay of Rose and O'Connell (9), in which NADP reduction is measured. In vivo inactivation of enzymes by paraquat. Yeast ceils (i0 mg/ml) were incubated with 0.i mm paraquat in i0 ml of 20 mm Tris-HC1 buffer (ph 7.5) at 37~ for 1 hr. The mixture was centrifuged at 3,000 rpm, and the yeast was suspended in 1 ml of 50 mm Tris-HC! containing 0.4 M sorbitol. The washed yeast ceils were permeabilized with toluene as described previously (10), and enzyme activity was determined in the permeabilized yeast celis. In situ inactivation of enzymes by copper plus ascorbic acid. Yeast cells (10 mg/ml) permeabilized with toluene (10) were incubated with 20 p31 CuC12, 2.5 mm ascorbic acid in 20 mm Tris-HCl buffer (ph 7.5) at 37~ for 1 hr. Enzyme activity was determined in yeast ceils collected by centrifugation at 3,000 rpm as described above. RESULTS We examined the effect of 02- production on some TCA cycle-related enzymes under the in vlvo conditions of yeast cells. Incubation of yeast cells with paraquat caused a specific inactivation of aconitase, but did not affect other TCA cycle-related enzymes including fumarase, NAD-, and NADP-linked isocitrate dehydrogenase, and malate dehydrogenase (Table 1). These results suggest that aconitase can be inactivated by superoxide radical produced by the reaction of 02 with paraquat radical formed. We further explored the effect of copper plus ascorbic acid on the TCA cycle enzymes in permeabilized yeast cells. Aconitase was markedly inactivated by copper plus ascorbate, whereas NADP-isocitrate dehydrogenase activity was not at all decreased. Fumarase and NAD-isocitrate dehydrogenase activities were decreased only a little (Table 2). 482

3 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL Table 1 Effect of paraquat on the activity of TCA cycle-related enzymes in yeast cells. Enzyme Treatment Activity (~mol/min per g yeast) Aconitase Control Paraquat 2.51 i 0.94 * Fumarase Control Paraquat NAD-linked Control Isocitrate dehydrogenase +Paraquat NADP-linked Control Isocitrate dehydrogenase +Paraquat Yeast cells (10 mg/ml) were incubated with 0.1 mm paraquat in 10 ml of 20 mm Tris-HC1 buffer (ph 7.5) at 37~ for 1 hr. After centrifugation the yeast was permeabilized with toluene as described previously (10), and used for determining the enzyme activity. Results are expressed as meanfsd of four experiments. The asterisk indicates p<0.001 relative to the control value. Table 2 Effect of copper plus ascorbate on the activity of TCA cycle-related enzymes in permeabilized yeast cells. Enzyme Treatment Activity (~mol/min per g yeast) Aconitase Control 4.69 i 0.44 Cu/Ascorbate * Fumarase Control Cu/Ascorbate NAD-linked Control Isocitrate dehydrogenase Cu/Ascorbate NADP-linked Control Isocitrate dehydrogenase Cu/Ascorbate Yeast cells (10 mg/ml) permeabilized with toluene (10) were incubated with 20 ~M CuC12, 2.5 film ascorbic acid in 20 n~i Tris-HC1 buffer (ph 7.5) at 37~ for 1 hr. Enzyme activity was determined in yeast cells collected by centrifugation at 3,000 rpm. Results are expressed as meanisd of four experiments. The asterisk indicates p<0.001 relative to the control value. 483

4 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Inactivation and its protection of aconitase was analyzed under the in situ conditions (Table 3). Paraquat, glucose 6-phosphate or NADP alone did not affect the aconitase activity; on the contrary, addition of paraquat with NADP and glucose 6-phosphate caused a marked inactivation of aconitase in permeabilized yeast. These results indicate that the inactivation of aconitase depends on the reduction of paraquat with NADPH formed by glucose 6-phosphate dehydrogenase (EC ). Inactivation of aeonitase was effectively prevented by the addition of citrate the substrate of aconitase. DISCUSSION Oxygen is an essential element for the terminal acceptor of the electrons during respiration the main source of energy in aerobic organisms. Molecular oxygen is reduced to water in electron transport system of mitochondria and microsomes. However, this process may produce some reactive oxygen species such as superoxide anion, hydrogen peroxide and hydroxyl radical as inevitable by-products. Superoxide radical, generated by oneelectron reduction of oxygen, serves as a source for secondary highly reactive hydroxyl radical (Ii), and has been incriminated as being the agent responsible for deleterious effects in many biological systems (12). Aconitase was readily inactivated by reactive oxygen species produced under the in vivo and in situ conditions of yeast cells. Treatment of yeast cells with paraquat can produce superoxide anion through the reaction of Table 3 Inactivation of aconitase by reduction of paraquat and its protection by addition of citrate in permeabilized yeast Addition Aconitase activity (~mol/min per g yeast) None mm Paraquat added 4.45 Paraquat plus 1 mm glucose 6-phosphate 3.50 Paraquat plus 10 gm NADP 3.10 Paraquat plus Glc-6-P + NADP mm Citrate plus paraquat + G6P + NADP 4,25 Yeast ceils (I0 mg/ml) permeabilized with toluene (i0) were incubated with the additives deseirbed above in 20 mm Tris-HCl buffer (ph 7.5) at 37~ for I0 min. Aconitase activity was determined in the permeabilized yeast cells. 484

5 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL oxygen with paraquat radical formed by the one-electron reduction of paraquat (13). This postulated mechanism was confirmed by the in si~u experiment which showed that the inactivation of aconitase required the enzymatic reduction of paraquat by NADP-cytochrome reductase (EC )(13) with NADPH formed through the action of glucose 6-phosphate dehydrogenase. Furthermore, addition of copper plus ascorbate effectively inactivated aconitase under the in situ conditions of permeabilized yeast cells. Cuprous ion formed by the reduction of cupric ion in the presence of ascorbate can reduce oxygen in the reaction mixture, resulting in the formation of superoxide anion. Superoxide radical inactivates aconitase which bear [4Fe-4S] prosthetic group: 02- releases labile iron from the [4Fe-4S] cluster of aconitase, giving a [3Fe-4S] cluster (14). The most sensitive target of the superoxide radical may be aconitase in yeast cells. On the other hand, citrate effectively prevented aconitase from the inactivation by paraquat. Citrate and fluorocitrate as the substrate and the competitive inhibitor of aconitase, respectively stabilize the [4Fe-4S] cluster of the enzyme active site (15). Thus, the increase in citrate can contribute to a protective mechanism against oxidative stress. Citrate concentrations in yeast largely depend on the growth conditions of cells: aerobically grown yeast contains 8 Imol of citrate/g fresh cells, which is decreased to 3 Imol/g when subjected to anaerobic conditions (16). This depletion of citrate has been considered to participate in the glycolytic stimulation by the reversal of the citrate inhibition of phosphofructokinase (EC ) in anaerobically grown yeast (16), and may be due to the increased activity of glutamate formation from citrate in anaerobiosis (17). Furthermore, protection by citrate of oxidative inactivation of aconitase suggests a novel physiological role of citrate in yeast. Aerobically grown yeast cells are exposed to highly oxidative stress, which can cause an inactivation of aconitase; however, aerobic yeast is resistant to oxygen toxicity. This mechanism can be explained by the increased concentrations of citrate in addition to the higher activity of superoxide dismutase under the aerobic conditions. The increase in citrate prevent aconitase from the inactivation in aerobic yeast. Furthermore, the oxidative inactivation of aconitase will cause an accumulation of citrate, which can, in turn, protect the enzyme from further inactivation. Increase in citrate levels of aerobic yeast may act as a defense mechanism to oxygen toxicity. 485

6 BIOCHEMISTRYondMOLECULAR BIOLOGY INTERNATIONAL REFERENCES i. Halliwell, B. and Gutteridge, J. M. C. (1984) Biochem. J. 219, Balzan, R., Bannister, W. H., Hunter, G. J. and Bannister, J. V. (1995) Proc. Natl. Acad. Sei. USA 92, McCord, J. M. and Fridovich, I. (1969) J. Biol. Chem. 244, McCord, J. M., Keele, B. B. Jr., and Fridovich, I. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, Gardner, P. R., and Fridovich, I. (1992) J. Biol. Chem. 267, Gardner, P. R., Nguyen, D.-D. H., and White, C. W. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, Yoshino, M. and Murakami, K. (1992) BioMetals 5, Racker, E. (1950) Biochim. Biophys. Acta 4, Rose, I. A. and O'Connell, E. L. (1967) J. Biol. Chem. 242, Murakami, K., Nagura, H., and Yoshino, M. (1980) Anal. Biochem. 105, Zer, H., Freedman, J. H., Peisach, J., and Chevion, M. (1991) Free Rad. Biol. Med. 11, Fridovich, I. (1986) Arch. Biochem. Biophys. 247, Tomita, M. (1991) Biochem. Pharmacol. 42, Flint, D. H., Tuminello, J. F. and Emptage, M. H. (1993) J. Biol. Chem. 288, Glusker, J. P. (1971) in The Enzymes (Boyer, P.D., ed) 3rd Ed., Vo].5, pp , Academic Press, New York. 16. Salas, M. L., Vinuela, E., Salas, M. and Sols, A. (1965) Biochem. Biophys. Res. Commun. 19, Yoshino, M. and Murakami, K. (1993) Int. J. Biochem. 25,

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